Eosinophil activation during immune responses: an ultrastructural view with an emphasis on viral diseases.

Eosinophils are cells of the innate immune system that orchestrate complex inflammatory responses. The study of the cell biology of eosinophils, particularly associated with cell activation, is of great interest to understand their immune responses. From a morphological perspective, activated eosinophils show ultrastructural signatures that have provided critical insights into the comprehension of their functional capabilities. Application of conventional transmission electron microscopy (TEM) in combination with quantitative assessments (quantitative TEM), molecular imaging (immunoEM), and three-dimensional (3D) electron tomography have generated important insights into mechanisms of eosinophil activation. This review explores a multitude of ultrastructural events taking place in eosinophils activated in vitro and in vivo as key players in allergic and inflammatory diseases, with an emphasis on viral infections. Recent progress in our understanding of biological processes underlying eosinophil activation, including in vivo mitochondrial remodeling, is discussed, and it can bring new thinking to the field.

Prevention of lipid droplet accumulation by DGAT1 inhibition ameliorates sepsis-induced liver injury and inflammation.

Background & Aims: Lipid droplet (LD) accumulation in cells and tissues is understood to be an evolutionarily conserved tissue tolerance mechanism to prevent lipotoxicity caused by excess lipids; however, the presence of excess LDs has been associated with numerous diseases. Sepsis triggers the reprogramming of lipid metabolism and LD accumulation in cells and tissues, including the liver. The functions and consequences of sepsis-triggered liver LD accumulation are not well known. Methods: Experimental sepsis was induced by CLP (caecal ligation and puncture) in mice. Markers of hepatic steatosis, liver injury, hepatic oxidative stress, and inflammation were analysed using a combination of functional, imaging, lipidomic, protein expression and immune-enzymatic assays. To prevent LD formation, mice were treated orally with A922500, a pharmacological inhibitor of DGAT1. Results: We identified that liver LD overload correlates with liver injury and sepsis severity. Moreover, the progression of steatosis from 24 h to 48 h post-CLP occurs in parallel with increased cytokine expression, inflammatory cell recruitment and oxidative stress. Lipidomic analysis of purified LDs demonstrated that sepsis leads LDs to harbour increased amounts of unsaturated fatty acids, mostly 18:1 and 18:2. An increased content of lipoperoxides within LDs was also observed. Conversely, the impairment of LD formation by inhibition of the DGAT1 enzyme reduces levels of hepatic inflammation and lipid peroxidation markers and ameliorates sepsis-induced liver injury. Conclusions: Our results indicate that sepsis triggers lipid metabolism alterations that culminate in increased liver LD accumulation. Increased LDs are associated with disease severity and liver injury. Moreover, inhibition of LD accumulation decreased the production of inflammatory mediators and lipid peroxidation while improving tissue function, suggesting that LDs contribute to the pathogenesis of liver injury triggered by sepsis. Impact and Implications: Sepsis is a complex life-threatening syndrome caused by dysregulated inflammatory and metabolic host responses to infection. The observation that lipid droplets may contribute to sepsis-associated organ injury by amplifying lipid peroxidation and inflammation provides a rationale for therapeutically targeting lipid droplets and lipid metabolism in sepsis.

Antibiofilm potential of Annona muricata L. ethanolic extract against multi-drug resistant Candida albicans.

ETNOPHARMACOLOGICAL RELEVANCE: Traditional uses of Annona muricata L. (soursop) include treatment for cancer, fungal infections, and inflammatory diseases. Its phytoconstituents, mainly acetogenins and alkaloids, are associated with therapeutic activity and clinical application is currently under investigation. However, the application of phytotherapy to treat diseases caused by fungal biofilms, such as vulvovaginal candidiasis (VVC), is still limited. AIM OF THE STUDY: To investigate the activity of the ethanolic extract of A. muricata leaves (AML) against biofilms formed by multiresistant Candida albicans (ATCC® 10231) both in vitro and in a VVC experimental model. MATERIAL AND METHODS: C. albicans biofilms were grown and their adhesion, proliferation, development, and matrix composition studied by spectrophotometry, scanning electron microscopy (SEM), whole slide imaging (WSI), and biochemical assays without or with AML treatment. In parallel, in vivo experiments were conducted using a murine model of infection treated with different concentrations of the extract and nystatin. Fungal burden and histological changes were investigated. RESULTS: The proliferation and adhesion of C. albicans biofilms were significantly reduced as confirmed by SEM and WSI quantitative analyses. Furthermore, the concentration of carbohydrates, proteins and DNA was reduced in the biofilm matrix. In vivo assays demonstrated that AML was able to reduce the fungal burden and the inflammatory process. CONCLUSIONS: The findings further emphasized the therapeutic and scientific potential of AML, thus encouraging its future use in the treatment of VVC.

Antifungal Annona muricata L. (soursop) extract targets the cell envelope of multi-drug resistant Candida albicans.

ETNOPHARMACOLOGICAL RELEVANCE: Annona muricata L. (soursop) is traditionally used in the treatment of inflammatory diseases, cancer, and infections caused by fungi. The therapeutic activity explored by its medicinal use is generally associated with its phytoconstituents, such as acetogenins and alkaloids. However, its potential antifungal bioactivity as well as its mechanism of action remains to be established. AIM OF THE STUDY: To evaluate the antifungal activity of the ethanolic extract of A. muricata leaves against multidrug-resistant Candida albicans (ATCC® 10231). MATERIAL AND METHODS: Phytoconstituents were detected by UFLC-QTOF-MS. The minimum inhibitory concentration was determined, followed by the determination of the minimum fungicidal concentration. For planktonic cells, the growth curve and cell density were evaluated. Studies to understand the mechanism of action on the cell envelope involved crystal violet permeability, membrane extravasation, sorbitol protection, exogenous ergosterol binding assay, metabolic activity, and cell viability. Furthermore, mitochondrial membrane potential was assessed. RESULTS: Our analyses demonstrated a significant inhibitory effect of A. muricata, with the ability to reduce fungal growth by 58% and cell density by 65%. The extract affected both the fungal plasma membrane and cell wall integrity, with significant reduction of the cell viability. Depolarization of the fungal mitochondrial membrane was observed after treatment with A. muricata. Rutin, xi-anomuricine, kaempferol-3O-rutinoside, nornuciferine, xylopine, atherosperminine, caffeic acid, asimilobine, s-norcorydine, loliolide, annohexocin, annomuricin, annopentocin, and sucrose were identified as extract bioactive components. CONCLUSIONS: Our findings show that the A. muricata extract is a source of chemical diversity, which acts as a potential antifungal agent with promising application to the therapy of infections caused by C. albicans.

Schistosomiasis Mansoni-Recruited Eosinophils: An Overview in the Granuloma Context.

Eosinophils are remarkably recruited during schistosomiasis mansoni, one of the most common parasitic diseases worldwide. These cells actively migrate and accumulate at sites of granulomatous inflammation termed granulomas, the main pathological feature of this disease. Eosinophils colonize granulomas as a robust cell population and establish complex interactions with other immune cells and with the granuloma microenvironment. Eosinophils are the most abundant cells in granulomas induced by Schistosoma mansoni infection, but their functions during this disease remain unclear and even controversial. Here, we explore the current information on eosinophils as components of Schistosoma mansoni granulomas in both humans and natural and experimental models and their potential significance as central cells triggered by this infection.

Enhanced ability of freshwater bacteria to secrete extracellular vesicles upon interaction with virus.

The ability of freshwater bacteria to secrete extracellular vesicles (EVs) upon interaction with viruses remains to be established. Here, we investigated for the first time if freshwater virus-infected bacteria release EVs in both natural ecosystems and virus-like particles (VLPs)-enriched cultures. We performed a systematic study using transmission electron microscopy to visualize viruses and EVs at high resolution and single-cell imaging analyses to quantitate nascent EVs at the surface of gram-negative bacteria. First, by analysing freshwater samples from a tropical ecosystem (Negro River/Amazon Basin/Brazil), we captured bacteriophages-infected bacteria releasing EVs from their outer membrane. Next, VLPs isolated from these samples and inoculated in bacterial cultures not only impacted bacteria growth and viability but also led them to a significant release of EVs (~300% increase in numbers/cell section) compared to controls. The numbers of both budding and free EVs and EVs per linear micrometre of cell envelope were significantly higher in infected bacteria. Our findings identify a yet-not recognized capability of freshwater bacteria in generating EVs (overvesiculation) in response to viral infection. Since viruses are abundant members of aquatic ecosystems and bacteria are natural hosts for them, such interaction is an interesting event for microbial communities to be explored in freshwater ecosystems.

Mitochondrial Population in Mouse Eosinophils: Ultrastructural Dynamics in Cell Differentiation and Inflammatory Diseases.

Mitochondria are multifunctional organelles of which ultrastructure is tightly linked to cell physiology. Accumulating evidence shows that mitochondrial remodeling has an impact on immune responses, but our current understanding of the mitochondrial architecture, interactions, and morphological changes in immune cells, mainly in eosinophils, is still poorly known. Here, we applied transmission electron microscopy (TEM), single-cell imaging analysis, and electron tomography, a technique that provides three-dimensional (3D) views at high resolution, to investigate mitochondrial dynamics in mouse eosinophils developing in cultures as well as in the context of inflammatory diseases characterized by recruitment and activation of these cells (mouse models of asthma, H1N1 influenza A virus (IAV) infection, and schistosomiasis mansoni). First, quantitative analyses showed that the mitochondrial area decrease 70% during eosinophil development (from undifferentiated precursor cells to mature eosinophils). Mitophagy, a consistent process revealed by TEM in immature but not in mature eosinophils, is likely operating in mitochondrial clearance during eosinophilopoiesis. Events of mitochondria interaction (inter-organelle membrane contacts) were also detected and quantitated within developing eosinophils and included mitochondria-endoplasmic reticulum, mitochondria-mitochondria, and mitochondria-secretory granules, all of them significantly higher in numbers in immature compared to mature cells. Moreover, single-mitochondrion analyses revealed that as the eosinophil matures, mitochondria cristae significantly increase in number and reshape to lamellar morphology. Eosinophils did not change (asthma) or reduced (IAV and Schistosoma infections) their mitochondrial mass in response to inflammatory diseases. However, asthma and schistosomiasis, but not IAV infection, induced amplification of both cristae numbers and volume in individual mitochondria. Mitochondrial cristae remodeling occurred in all inflammatory conditions with the proportions of mitochondria containing only lamellar or tubular, or mixed cristae (an ultrastructural aspect seen just in tissue eosinophils) depending on the tissue/disease microenvironment. The ability of mitochondria to interact with granules, mainly mobilized ones, was remarkably captured by TEM in eosinophils participating in all inflammatory diseases. Altogether, we demonstrate that the processes of eosinophilopoiesis and inflammation-induced activation interfere with the mitochondrial dynamics within mouse eosinophils leading to cristae remodeling and inter-organelle contacts. The understanding of how mitochondrial dynamics contribute to eosinophil immune functions is an open interesting field to be explored.

Global RNAseq of ocular cells reveals gene dysregulation in both asymptomatic and with Congenital Zika Syndrome infants exposed prenatally to Zika virus.

In 2015, Brazil reported an outbreak identified as Zika virus (ZIKV) infection associated with congenital abnormalities. To date, a total of 86 countries and territories have described evidence of Zika infection and recently the appearance of the African ZIKV lineage in Brazil highlights the risk of a new epidemic. The spectrum of ZIKV infection-induced alterations at both cellular and molecular levels is not completely elucidated. Here, we present for the first time the gene expression responses associated with prenatal ZIKV infection from ocular cells. We applied a recently developed non-invasive method (impression cytology) which use eye cells as a model for ZIKV studies. The ocular profiling revealed significant differences between exposed and control groups, as well as a different pattern in ocular transcripts from Congenital Zika Syndrome (CZS) compared to ZIKV-exposed but asymptomatic infants. Our data showed pathways related to mismatch repair, cancer, and PI3K/AKT/mTOR signaling and genes probably causative or protective in the modulation of ZIKV infection. Ocular cells revealed the effects of ZIKV infection on primordial neuronal cell genes, evidenced by changes in genes associated with embryonic cells. The changes in gene expression support an association with the gestational period of the infection and provide evidence for the resulting clinical and ophthalmological pathologies. Additionally, the findings of cell death- and cancer-associated deregulated genes raise concerns about the early onset of other potential pathologies including the need for tumor surveillance. Our results thus provide direct evidence that infants exposed prenatally to the Zika virus, not only with CZS but also without clinical signs (asymptomatic) express cellular and molecular changes with potential clinical implications.

Functionalized 1,2,3-triazolium salts as potential agents against visceral leishmaniasis.

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, being fatal if untreated. In search of a more effective treatment for VL, one of the main strategies is the development and screening of new antileishmanial compounds. Here, we reported the synthesis of seven new acetyl functionalized 1,2,3-triazolium salts, together with four 1,2,3-triazole precursors, and investigated their effect against different strains of L. infantum from dogs and humans. The 1,2,3-triazolium salts exhibited better activity than the 1,2,3-triazole derivatives with IC50 range from 0.12 to 8.66 µM and, among them, compound 5 showed significant activity against promastigotes (IC50 from 4.55 to 5.28 µM) and intracellular amastigotes (IC50 from 5.36 to 7.92 µM), with the best selective index (SI ~ 6-9) and reduced toxicity. Our findings, using biochemical and ultrastructural approaches, demonstrated that compound 5 targets the mitochondrion of L. infantum promastigotes, leading to the formation of reactive oxygen species (ROS), increase of the mitochondrial membrane potential, and mitochondrial alteration. Moreover, quantitative transmission electron microscopy (TEM) revealed that compound 5 induces the reduction of promastigote size and cytoplasmic vacuolization. Interestingly, the effect of compound 5 was not associated with apoptosis or necrosis of the parasites but, instead, seems to be mediated through a pathway involving autophagy, with a clear detection of autophagic vacuoles in the cytoplasm by using both a fluorescent marker and TEM. As for the in vivo studies, compound 5 showed activity in a mouse model of VL at 20 mg/kg, reducing the parasite load in both spleen and liver (59.80% and 26.88%, respectively). Finally, this compound did not induce hepatoxicity or nephrotoxicity and was able to normalize the altered biochemical parameters in the infected mice. Thus, our findings support the use of 1,2,3-triazolium salts as potential agents against visceral leishmaniasis.

Spilanthol as a promising antifungal alkylamide for the treatment of vulvovaginal candidiasis.

Spilanthol is a bioactive alkylamide from the native Amazon plant species, Acmella oleracea. However, antifungal activities of spilanthol and its application to the therapeutic treatment of candidiasis remain to be explored. This study sought to evaluate the in vitro and in vivo antifungal activity of spilanthol previously isolated from A. oleracea (spilanthol(AcO)) against Candida albicans ATCC® 10231™, a multidrug-resistant fungal strain. Microdilution methods were used to determine inhibitory and fungicidal concentrations of spilanthol(AcO). In planktonic cultures, the fungal growth kinetics, yeast cell metabolic activity, cell membrane permeability and cell wall integrity were investigated. The effect of spilanthol(AcO) on the proliferation and adhesion of fungal biofilms was evaluated by whole slide imaging and scanning electron microscopy. The biochemical composition of the biofilm matrix was also analyzed. In parallel, spilanthol(AcO) was tested in vivo in an experimental vulvovaginal candidiasis model. Our in vitro analyses in C. albicans planktonic cultures detected a significant inhibitory effect of spilanthol(AcO), which affects both yeast cell membrane and cell wall integrity, interfering with the fungus growth. C. albicans biofilm proliferation and adhesion, as well as, carbohydrates and DNA in biofilm matrix were reduced after spilanthol(AcO) treatment. Moreover, infected rats treated with spilanthol(AcO) showed consistent reduction of both fungal burden and inflammatory processes compared to the untreated animals. Altogether, our findings demonstrated that spilanthol(AcO) is an bioactive compound against planktonic and biofilm forms of a multidrug resistant C. albicans strain. Furthermore, spilanthol(AcO) can be potentially considered for therapeutical treatment of vulvovaginal candidiasis caused by C. albicans. LAY SUMMARY: This study sought to evaluate the antifungal activity of spilanthol against Candida albicans ATCC® 10 231™, a multidrug-resistant fungal strain. Our findings demonstrated that spilanthol(AcO) can be potentially considered for therapeutical treatment of vulvovaginal candidiasis caused by C. albicans.

Whole slide imaging is a high-throughput method to assess Candida biofilm formation.

New strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyse fungal biofilm growth.

Changing our view of the Schistosoma granuloma to an ecological standpoint.

Schistosomiasis, a neglected parasitic tropical disease that has plagued humans for centuries, remains a major public health burden. A primary challenge to understanding schistosomiasis is deciphering the most remarkable pathological feature of this disease, the granuloma - a highly dynamic and self-organized structure formed by both host and parasite components. Granulomas are considered a remarkable example of how parasites evolved with their hosts to establish complex and intimate associations. However, much remains unclear regarding life within the granuloma, and strategies to restrain its development are still lacking. Here we explore current information on the hepatic Schistosoma mansoni granuloma in the light of Ecology and propose that this intricate structure acts as a real ecosystem. The schistosomal granuloma is formed by cells (biotic component), protein scaffolds, fibres, and chemical compounds (abiotic components) with inputs/outputs of energy and matter, as complex as in classical ecosystems. We review the distinct cell populations ('species') within the granuloma and examine how they integrate with each other and interact with their microenvironment to form a multifaceted cell community in different space-time frames. The colonization of the hepatic tissue to form granulomas is explained from the point of view of an ecological succession whereby a community is able to modify its physical environment, creating conditions and resources for ecosystem construction. Remarkably, the granuloma represents a dynamic evolutionary system that undergoes progressive changes in the 'species' that compose its community over time. In line with ecological concepts, we examine the granuloma not only as a place where a community of cells is settled (spatial niche or habitat) but also as a site in which the functional activities of these combined populations occur in an orchestrated way in response to microenvironmental gradients such as cytokines and egg antigens. Finally, we assert how the levels of organization of cellular components in a granuloma as conventionally defined by Cell Biology can fit perfectly into a hierarchical structure of biological systems as defined by Ecology. By rethinking the granuloma as an integrating and evolving ecosystem, we draw attention to the inner workings of this structure that are central to the understanding of schistosomiasis and could guide its future treatment.

Antifungal Activity of the Natural Coumarin Scopoletin Against Planktonic Cells and Biofilms From a Multidrug-Resistant Candida tropicalis Strain.

Candida tropicalis is one the most relevant biofilm-forming fungal species increasingly associated with invasive mucosal candidiasis worldwide. The amplified antifungal resistance supports the necessity for more effective and less toxic treatment, including the use of plant-derived natural products. Scopoletin, a natural coumarin, has shown antifungal properties against plant yeast pathogens. However, the antifungal activity of this coumarin against clinically relevant fungal species such as C. tropicalis remains to be established. Here, we investigated the potential antifungal properties and mechanisms of action of scopoletin against a multidrug-resistant C. tropicalis strain (ATCC 28707). First, scopoletin was isolated by high-performance liquid chromatography from Mitracarpus frigidus, a plant species (family Rubiaceae) distributed throughout South America. Next, scopoletin was tested on C. tropicalis cultivated for 48h in both planktonic and biofilm forms. Fungal planktonic growth inhibition was analyzed by evaluating minimal inhibitory concentration (MIC), time-kill kinetics and cell density whereas the mechanisms of action were investigated with nucleotide leakage, efflux pumps and sorbitol and ergosterol bioassays. Finally, the scopoletin ability to affect C. tropicalis biofilms was evaluated through spectrophotometric and whole slide imaging approaches. In all procedures, fluconazole was used as a positive control. MIC values for scopoletin and fluconazole were 50 and 250 µg/L respectively, thus demonstrating a fungistatic activity for scopoletin. Scopoletin induced a significant decrease of C. tropicalis growth curves and cell density (91.7% reduction) compared to the growth control. Its action was related to the fungal cell wall, affecting plasma membrane sterols. When associated with fluconazole, scopoletin led to inhibition of efflux pumps at the plasma membrane. Moreover, scopoletin not only inhibited the growth rate of preformed biofilms (68.2% inhibition at MIC value) but also significantly decreased the extent of biofilms growing on the surface of coverslips, preventing the formation of elongated fungal forms. Our data demonstrate, for the first time, that scopoletin act as an effective antifungal phytocompound against a multidrug-resistant strain of C. tropicalis with properties that affect both planktonic and biofilm forms of this pathogen. Thus, the present findings support additional studies for antifungal drug development based on plant isolated-scopoletin to treat candidiasis caused by C. tropicalis.

Cellular Imprinting Proteomics Assay: A Novel Method for Detection of Neural and Ocular Disorders Applied to Congenital Zika Virus Syndrome.

Congenital Zika syndrome was first described due to increased incidence of congenital abnormalities associated with Zika virus (ZIKV) infection. Since the eye develops as part of the embryo central nervous system (CNS) structure, it becomes a specialized compartment able to display symptoms of neurodegenerative diseases and has been proposed as a noninvasive approach to the early diagnosis of neurological diseases. Ocular lesions result from defects that occurred during embryogenesis and can become apparent in newborns exposed to ZIKV. Furthermore, the absence of microcephaly cannot exclude the occurrence of ocular lesions and other CNS manifestations. Considering the need for surveillance of newborns and infants with possible congenital exposure, we developed a method termed cellular imprinting proteomic assay (CImPA) to evaluate the ocular surface proteome specific to infants exposed to ZIKV during gestation compared to nonexposure. CImPA combines surface cells and fluid capture using membrane disks and a large-scale quantitative proteomics approach, which allowed the first-time report of molecular alterations such as neutrophil degranulation, cell death signaling, ocular and neurological pathways, which are associated with ZIKV infection with and without the development of congenital Zika syndrome, CZS. Particularly, infants exposed to ZIKV during gestation and without early clinical symptoms could be detected using the CImPA method. Lastly, this methodology has broad applicability as it could be translated in the study of several neurological diseases to identify novel diagnostic biomarkers. Data are available via ProteomeXchange with identifier PXD014038.

Galectin-10, the protein that forms Charcot-Leyden crystals, is not stored in granules but resides in the peripheral cytoplasm of human eosinophils.

A predominant protein of human eosinophils is galectin-10 (Gal-10), also known as Charcot-Leyden crystal protein (CLC-P) because of its remarkable ability to form Charcot-Leyden crystals (CLCs), which are frequently found in tissues from patients with eosinophilic disorders. CLC-P/Gal-10 is highly expressed in human eosinophils and considered a biomarker of eosinophil involvement in inflammation. However, the intracellular sites where large pools of CLC-P/Gal-10 constitutively reside are still unclear, and whether this protein is derived or not from eosinophil granules remains to be established. Here, we applied pre-embedding immunonanogold transmission electron microscopy combined with strategies for optimal antigen and cell preservation and quantitative imaging analysis to investigate, for the first time, the intracellular localization of CLC-P/Gal-10 at high resolution in resting and activated human eosinophils. We demonstrated that CLC-P/Gal-10 is mostly stored in the peripheral cytoplasm of human eosinophils, being accumulated within an area of ∼250 nm wide underneath the plasma membrane and not within specific (secretory) granules, a pattern also observed by immunofluorescence. High-resolution analysis of single cells revealed that CLC-P/Gal-10 interacts with the plasma membrane with immunoreactive microdomains of high CLC-P/Gal-10 density being found in ∼60% of the membrane area. Eosinophil stimulation with CCL11 or TNF-α, which are known inducers of eosinophil secretion, did not change the peripheral localization of CLC-P/Gal-10 as observed by both immunofluorescence and immuno-EM (electron microscopy). Thus, in contrast to other preformed eosinophil proteins, CLC-P/Gal-10 neither is stored within secretory granules nor exported through classical degranulation mechanisms (piecemeal degranulation and compound exocytosis).

Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies.

IMPORTANCE: Non-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined. OBJECTIVES: The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches. DESIGN SETTINGS AND PARTICIPANTS: Ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments. RESULTS: Our optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies. CONCLUSIONS AND RELEVANCE: Our method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.

Whole Slide Imaging and Its Applications to Histopathological Studies of Liver Disorders.

Histological analysis of hepatic tissue specimens is essential for evaluating the pathology of several liver disorders such as chronic liver diseases, hepatocellular carcinomas, liver steatosis, and infectious liver diseases. Manual examination of histological slides on the microscope is a classically used method to study these disorders. However, it is considered time-consuming, limited, and associated with intra- and inter-observer variability. Emerging technologies such as whole slide imaging (WSI), also termed virtual microscopy, have increasingly been used to improve the assessment of histological features with applications in both clinical and research laboratories. WSI enables the acquisition of the tissue morphology/pathology from glass slides and translates it into a digital form comparable to a conventional microscope, but with several advantages such as easy image accessibility and storage, portability, sharing, annotation, qualitative and quantitative image analysis, and use for educational purposes. WSI-generated images simultaneously provide high resolution and a wide field of observation that can cover the entire section, extending any single field of view. In this review, we summarize current knowledge on the application of WSI to histopathological analyses of liver disorders as well as to understand liver biology. We address how WSI may improve the assessment and quantification of multiple histological parameters in the liver, and help diagnose several hepatic conditions with important clinical implications. The WSI technical limitations are also discussed.

Antibacterial and Antibiofilm Activities of Psychorubrin, a Pyranonaphthoquinone Isolated From Mitracarpus frigidus (Rubiaceae).

Psychorubrin, a natural pyranonaphthoquinone found in different plants, has become an interesting compound in the search for new antimicrobial therapeutic agents. Here, we investigated the potential antagonistic activity of psychorubrin against planktonic and biofilm bacteria. First, psychorubrin was tested against several Gram-positive and Gram-negative bacteria strains by a broth microdilution susceptibility method. Second, bacterial killing assay, bacterial abundance, and membrane viability were evaluated. The nucleotide leakage assay was used to verify membrane destabilization while antibiofilm activities were analyzed by the effect on established biofilm, static biofilm formation, isolation of biofilm matrix assay and scanning electron microscopy. In parallel, the combinatorial effect of psychorubrin and chloramphenicol was evaluated by the checkerboard method. Psychorubrin was active against Gram-positive bacteria, showing rapid time-dependent kinetics of bacterial killing, amplified nucleotide leakage, and greater activity against the methicillin-resistant species (MRSA) Staphylococcus aureus 33591 and 33592 and Staphylococcus pyogenes 10096. Psychorubrin also interfered with the composition of the biofilm matrix by reducing the total content of carbohydrates and proteins. A synergic effect between psychorubrin and chloramphenicol was observed for S. aureus 33592 and S. pyogenes 10096 while an additive effect was detected for S. aureus 33591. Our findings demonstrate, for the first time, an antagonistic activity of psychorubrin against bacteria not only in their planktonic forms but also in biofilms, and identify bacterial membranes as primary targets for this compound. Based on these observations, psychorubrin has a good potential for the design of novel antimicrobial agents.

The Cyanobacterium Cylindrospermopsis raciborskii (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution.

Secretion of membrane-limited vesicles, collectively termed extracellular vesicles (EVs), is an important biological process of both eukaryotic and prokaryotic cells. This process has been observed in bacteria, but remains to be better characterized at high resolution in cyanobacteria. In the present work, we address the release of EVs by Cylindrospermopsis raciborskii (CYRF-01), a filamentous bloom-forming cyanobacterium, exposed to environmental stressors. First, non-axenic cultures of C. raciborskii (CYRF-01) were exposed to ultraviolet radiation (UVA + UVB) over a 6 h period, which is known to induce structural damage to this species. Second, C. raciborskii was co-cultured in interaction with another cyanobacterium species, Microcystis aeruginosa (MIRF-01), over a 24 h period. After the incubation times, cell density and viability were analyzed, and samples were processed for transmission electron microscopy (TEM). Our ultrastructural analyses revealed that C. raciborskii constitutively releases EVs from the outer membrane during its normal growth and amplifies such ability in response to environmental stressors. Both situations induced significant formation of outer membrane vesicles (OMVs) by C. raciborskii compared to control cells. Quantitative TEM revealed an increase of 48% (UV) and 60% (interaction) in the OMV numbers compared to control groups. Considering all groups, the OMVs ranged in size from 20 to 300 nm in diameter, with most OMVs showing diameters between 20 and 140 nm. Additionally, we detected that OMV formation is accompanied by phosphatidylserine exposure, a molecular event also observed in EV-secreting eukaryotic cells. Altogether, we identified for the first time that C. raciborskii has the competence to secrete OMVs and that under different stress situations the genesis of these vesicles is increased. The amplified ability of cyanobacteria to release OMVs may be associated with adaptive responses to changes in environmental conditions and interspecies cell communication.

Identification of Piecemeal Degranulation and Vesicular Transport of MBP-1 in Liver-Infiltrating Mouse Eosinophils During Acute Experimental Schistosoma mansoni Infection.

Eosinophils have been long associated with helminthic infections, although their functions in these diseases remain unclear. During schistosomiasis caused by the trematode Schistosoma mansoni, eosinophils are specifically recruited and migrate to sites of granulomatous responses where they degranulate. However, little is known about the mechanisms of eosinophil secretion during this disease. Here, we investigated the degranulation patterns, including the cellular mechanisms of major basic protein-1 (MBP-1) release, from inflammatory eosinophils in a mouse model of S. mansoni infection (acute phase). Fragments of the liver, a major target organ of this disease, were processed for histologic analyses (whole slide imaging), conventional transmission electron microscopy (TEM), and immunonanogold EM using a pre-embedding approach for precise localization of major basic protein 1 (MBP-1), a typical cationic protein stored pre-synthesized in eosinophil secretory (specific) granules. A well-characterized granulomatous inflammatory response with a high number of infiltrating eosinophils surrounding S. mansoni eggs was observed in the livers of infected mice. Moreover, significant elevations in the levels of plasma Th2 cytokines (IL-4, IL-13, and IL-10) and serum enzymes (alanine aminotransferase and aspartate aminotransferase) reflecting altered liver function were detected in response to the infection. TEM quantitative analyses revealed that while 19.1% of eosinophils were intact, most of them showed distinct degranulation processes: cytolysis (13.0%), classical and/or compound exocytosis identified by granule fusions (1.5%), and mainly piecemeal degranulation (PMD) (66.4%), which is mediated by vesicular trafficking. Immunonanogold EM showed a consistent labeling for MBP-1 associated with secretory granules. Most MBP-1-positive granules had PMD features (79.0 ± 4.8%). MBP-1 was also present extracellularly and on vesicles distributed in the cytoplasm and attached to/surrounding the surface of emptying granules. Our data demonstrated that liver-infiltrating mouse eosinophils are able to degranulate through different secretory processes during acute experimental S. mansoni infections with PMD being the predominant mechanism of eosinophil secretion. This means that a selective secretion of MBP-1 is occurring. Moreover, our study demonstrates, for the first time, a vesicular trafficking of MBP-1 within mouse eosinophils elicited by a helminth infection. Vesicle-mediated secretion of MBP-1 may be relevant for the rapid release of small concentrations of MBP-1 under cell activation.